The present invention relates to a method for determining the amount of a hapten present in a sample by the utilization of antigen-antibody reaction. In particular, it relates to a method for determining a hapten which may be utilized as a clinical analysis such as a serum analysis for determining the blood level of hormones or drugs in a field of medical diagnosis.
A low-molecular compound, which itself is not capable of raising antibody production in a living body, but acts as an antigen and becomes capable of raising antibody production when bound to a high-molecular compound such as protein, is referred to as a hapten. In a field of clinical analysis, immunochemical determination of the concentration of various substances such as low-molecular hormones and drugs in blood is practiced by the utilization of the antibodies obtained in the manner described above using the substances as haptens.
Radioimmunoassay (RIA), enzyme immunoassay (EIA) and fluorescence polarization immunoassay can be cited as principle examples of immunochemical determination techniques for hapten. This classification of the techniques depend on the difference of the labels by which the extent of reaction is determined. From the aspect of operating procedure, the techniques can be classified into so-called B/F separation in which determination is made by separating the reacted (bound) form and the unreacted (free) form of the labeled antigen or labeled antibody, and so-called homogeneous method in which no such separation is required.
The B/F separation method is employed in a part of RIA and EIA. This method is sensitive and specific, but has the defect that it is tedious in operation and requires a long time period for determination, because it involves troublesome separation-washing operations. The increase in frequency of handling a sample during the operations such as separation, washing, etc., leads to a greater risk to infection from the sample. In addition, RIA involves many difficult problems in handling such as the problem of disposal of radioactive wastes and necessity for specific equipments. EIA, because of the use of an enzyme as the label, has the disadvantages that strict control is required for reaction time and temperature, and that the determination tends to be affected by inhibition reactions.
The homogeneous method is employed in fluorescence polarization immunoassay and a part of EIA. This method is simple in operation, relatively short in time required for determination and easy to automate by the utilization of a particular device. However, because of low sensitivity, this method is limited to determination of certain types of drugs and is difficult to determine the substances present in a level of ng/ml such as many types of hormones and digoxin. Further, fluorescence polarization determination requires an expensive apparatus. The homogeneous EIA also has the disadvantages inherent in a method using an enzyme, and it may be said that inhibition reactions are unavoidable in this method because the substances to be determined remains in the system.
Latex agglutination is also a type of homogeneous method. This method is used for determination of a certain type of hapten mainly in a manner of slide method which is a qualitative or semi-quantitative method. In addition to the advantages of homogeneous method, i.e., simple operation and a short determination time, this method has the merit of being excellent in reagent storage stability and stability of reaction system as compared with EIA and other assay methods. However, low detection sensitivity is the greatest defect of this method (Japanese Patent Application Laid-Open (KOKAI) Nos. 53-104726(1978), 55-52945(1980) , 55-52946(1980), 55-156866(1980) and 61-265571(1986)).
A method for immunochemical determination of a hapten utilizing latex agglutination as described above, i.e., a method in which the extent of inhibition by hapten against the agglutination of latex particles through a high-molecular compound to which a hapten is bound is determined, has problems in sensitivity. One problem is difficulty to obtain a high titer antibody to a hapten, especially one derived from a living body, while an antibody with a high titer is necessary for effecting the latex agglutination. Another problem is that it is difficult to improve inhibition sensitivity from the methodological reason involved in the method, i.e., the necessity of covering a large portion of an antibody with a hapten for inhibiting the agglutination whereas the antibody present in only a part on the latex particle surface is sufficient for agglutination.
WO 89/01161 discloses a method for determining the concentration of a hapten in a sample, which comprises the steps of:
incubating a mixture of the sample containing the hapten with first magnetically attractable particles carrying the hapten and second particles carrying an antibody to the hapten,
applying a magnetic field to the mixture to bring down unreacted first particles and agglutinated particles of the first and second particles, and
measuring the optical density of the mixture, thereby determining the concentration of the hapten in the sample from the decrease in turbidity. In this method, also, an antibody with a high titer to the hapten is required, and therefore, a method which is highly sensitive and capable of using an antibody with a lower titer to a hapten has been desired.